RESUMEN
We report that a single growth factor, NM23-H1, enables serial passaging of both human ES and iPS cells in the absence of feeder cells, their conditioned media or bFGF in a fully defined xeno-free media on a novel defined, xeno-free surface. Stem cells cultured in this system show a gene expression pattern indicative of a more "naïve" state than stem cells grown in bFGF-based media. NM23-H1 and MUC1* growth factor receptor cooperate to control stem cell self-replication. By manipulating the multimerization state of NM23-H1, we override the stem cell's inherent programming that turns off pluripotency and trick the cells into continuously replicating as pluripotent stem cells. Dimeric NM23-H1 binds to and dimerizes the extra cellular domain of the MUC1* transmembrane receptor which stimulates growth and promotes pluripotency. Inhibition of the NM23-H1/MUC1* interaction accelerates differentiation and causes a spike in miR-145 expression which signals a cell's exit from pluripotency.
Asunto(s)
Nucleósido Difosfato Quinasas NM23/farmacología , Células Madre/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Unión Competitiva , Biomarcadores/metabolismo , Diferenciación Celular , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ligandos , MicroARNs/genética , MicroARNs/metabolismo , Mucina-1/inmunología , Mucina-1/metabolismo , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/metabolismo , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Células Madre/citología , Células Madre/metabolismoRESUMEN
A novel series of 4-aminophenylalanine and 4-aminocyclohexylalanine derivatives were designed and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4). The phenylalanine series afforded compounds such as 10 that were potent and selective (DPP-4, IC(50)=28nM), but exhibited limited oral bioavailability. The corresponding cyclohexylalanine derivatives such as 25 afforded improved PK exposure and efficacy in a murine OGTT experiment. The X-ray crystal structure of 25 bound to the DPP-4 active site is presented.
Asunto(s)
Alanina/análogos & derivados , Ciclohexanos/farmacología , Inhibidores de la Dipeptidil-Peptidasa IV , Inhibidores Enzimáticos/farmacología , Fenilalanina/análogos & derivados , Administración Oral , Alanina/química , Alanina/farmacología , Animales , Sitios de Unión , Disponibilidad Biológica , Cristalografía por Rayos X , Ciclohexanos/química , Dipeptidil Peptidasa 4/química , Inhibidores Enzimáticos/química , Prueba de Tolerancia a la Glucosa , Ratones , Modelos Moleculares , Estructura Molecular , Fenilalanina/química , Fenilalanina/farmacología , Relación Estructura-ActividadRESUMEN
The demonstration of pharmacodynamic efficacy of novel chemical entities represents a formidable challenge in the early exploration of synthetic lead classes. Here, we demonstrate a technique to validate the biological efficacy of novel antagonists of the human glucagon receptor (hGCGR) in the surgically removed perfused liver prior to the optimization of the pharmacokinetic properties of the compounds. The technique involves the direct observation by (13)C NMR of the biosynthesis of [(13)C]glycogen from [(13)C]pyruvate via the gluconeogenic pathway. The rapid breakdown of [(13)C]glycogen (glycogenolysis) following the addition of 50 pM exogenous glucagon is then monitored in real time in the perfused liver by (13)C NMR. The concentration-dependent inhibition of glucagon-mediated glycogenolysis is demonstrated for both the peptidyl glucagon receptor antagonist 1 and structurally diverse synthetic antagonists 2-7. Perfused livers were obtained from a transgenic mouse strain that exclusively expresses the functional human glucagon receptor, conferring human relevance to the activity observed with glucagon receptor antagonists. This technique does not provide adequate quantitative precision for the comparative ranking of active compounds, but does afford physiological evidence of efficacy in the early development of a chemical series of antagonists.
Asunto(s)
Hígado/metabolismo , Receptores de Glucagón/antagonistas & inhibidores , Animales , Células CHO , Radioisótopos de Carbono , Cricetinae , Humanos , Glucógeno Hepático/biosíntesis , Espectroscopía de Resonancia Magnética/métodos , Masculino , Ratones , Ratones Transgénicos , Estructura Molecular , Ácido Pirúvico/metabolismo , Receptores de Glucagón/metabolismo , Factores de TiempoRESUMEN
A novel class of antagonists of the human glucagon receptor (hGCGR) has been discovered. Systematic modification of the lead compound identified substituents that were essential for activity and those that were amenable to further optimization. This SAR exploration resulted in the synthesis of 13, which exhibited good potency as an hGCGR functional antagonist (IC50 = 34 nM) and moderate bioavailability (36% in mice).
Asunto(s)
Receptores de Glucagón/antagonistas & inhibidores , Tiofenos/síntesis química , Tiofenos/farmacología , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tiofenos/clasificaciónRESUMEN
HIV-1 protease inhibitors (PI) with an N-arylpyrrole moiety in the P(3) position afforded excellent antiviral potency and substantially improved aqueous solubility over previously reported variants. The rapid in vitro clearance of these compounds in human liver microsomes prompted oral coadministration with indinavir to hinder their metabolism by the cyctochrome P450 3A4 isozyme and allow for in vivo PK assessment.
Asunto(s)
Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/metabolismo , VIH-1/efectos de los fármacos , Indinavir/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Administración Oral , Biotransformación , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Diseño de Fármacos , Farmacorresistencia Viral , Quimioterapia Combinada , Inhibidores de la Proteasa del VIH/farmacocinética , Humanos , Indinavir/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Pirroles/síntesis química , Pirroles/farmacología , Relación Estructura-ActividadRESUMEN
Transposition of the pyridyl nitrogen from the P(3) substituent to the P(1)' substituent in HIV-1 protease inhibitors (PI) affords compounds such as 3 with an improved inhibitory profile against multiple P450 isoforms. These compounds also displayed increased potency, with 3 inhibiting viral spread (CIC(95)) at <8 nM for every strain of PI-resistant HIV-1 tested. The poor to modest bioavailability of these compounds may correlate in part to their aqueous solubility.
Asunto(s)
Inhibidores de la Proteasa del VIH/química , Proteasa del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/enzimología , Animales , Perros , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/fisiología , Inhibidores de la Proteasa del VIH/administración & dosificación , HumanosRESUMEN
Substitution of the t-butylcarboxamide substituent in analogues of the HIV protease inhibitor (PI) Indinavir with a trifluoroethylamide moiety confers greater potency against both the wild-type (NL4-3) virus and PI-resistant HIV. The trifluoroethyl substituent also affords a slower clearance rate in vivo (dogs); however, this may be due to more potent inhibition of at least two P450 isoforms.